The theme of this proposal is to understand the function of CTLA-4 (CD152) in T cell immunobiology and to elucidate the molecular mechanisms responsible for these processes. CTLA-4 is emerging as a key regulator of peripheral T cell activation and homeostasis. While it is clear that CTLA-4 ligation inhibits T cell responses, the time(s) during T cell activation and expansion and the mechanisms involved are largely unknown. The objective of this proposal is to understand when and how CTLA-4 inhibits T cell activation. The specific aims of this proposal are: (1) to test the hypothesis that CTLA-4 ligation modulates T cell responses by increasing the 'cumulative threshold' necessary for T cell activation. We postulate that CTLA-4ligation functions to increase the 'cumulative threshold' necessary for T cell activation. Based on this hypothesis we predict that the duration of T celI-APC interaction necessary for full T cell activation is increased in the presence of CTLA-4 ligation. We will test this prediction in Aim 1. (2) to test the prediction that CTLA-4 ligation during the T-APC interaction not only regulates the magnitude of the primary T cell response, but that CTLA-4 ligation alters the ability of the T cell to respond to subsequent stimulation. We postulate that this is due to the induction of additional negative signals. In Specific Aim 2 we will test these predictions and will test several candidate proteins as potential downstream mediators of CTLA-4 function; and (3) to elucidate protein-protein interactions involved in CTLA-4-mediated inhibition. The experimental approach is to screen for novel proteins associated with CTLA-4 cytoplasmic tail. We will test these candidate proteins in vitro and in vivo using the candidate genes identified in the 35S-labelled proteins associated with the CTLA-4cyt tail. We will also test the functional relevance of the proteins reported to bind to CTLA-4. To do this we will use RNA interference (siRNA) to specifically down modulate the expression of the candidate proteins in an inducible CTLA-4 T cell line. The insights gained from the proposed studies will permit a better definition of the mechanistic basis of the CTLA-4-mediated regulation of T cell responsiveness, T cell tolerance and loss of tolerance leading to autoimmunity. Understanding these processes is critical for designing novel therapeutic approaches for vaccine development, autoimmune disease, and in transplant and tumor immunology.